2013 AMBER Tutorial with UMP and OMP

In this tutorial, we will learn how to run a molecular dynamics simulation of a protein-ligand complex. We will then post-process that simulation by calculating structural fluctuations (with RMSD) and free energies of binding (MM-GBSA).

I. Introduction

AMBER

Amber - Assisted Model Building with Energy Refinement - is a suite of about multiple programs for perform macromolecular simulations. Amber11, the current version of Amber, includes newly released functionality such as PMEMD, particle mesh Ewald MD and soft-core Thermodynamics Integration MD. For the tutorial, we are using the newest version AMBER12.

The Amber 12 Manual is the primary resource to get started with Amber12. (Tip: Using Adobe Acrobat to view the file, you can simply search the document for keywords such as the name of a simulation parameter, which saves much time.) In addition, Amber Tools User's Manual serves as another reference while using Amber tools.

Here are some programs in Amber

LEaP: an preparing program for constructing new or modified systems in Amber. It consists of the functions of prep, link, edit, and parm for earlier version of Amber.

ANTECHAMBER: in additional to LEap, this main Antechamber suite program is for preparing input files other than standard nucleic acids and proteins.

SANDER: according to the Amber 12 manual, it is 'a basic energy minimizer and molecular dynamics program' that can be used to minimize, equilibrate and sample molecular conformations. And this is the program we mainly use in this tutorial to generate trajectory files of the molecular system.

PMEMD: version of SANDER that has improved parallel scaling property and optimized speed.

PTRAJ: an analysis program for processing trajectory files. One can use ptraj to rotate, translate the structures, evaluate geometrical features and so on.

There is a mailing list you could sign-up for, as an additional resource.

UMP and OMP

Organizing Directories

While performing MD simulations, it is convenient to adopt a standard directory structure / naming scheme, so that files are easy to find / identify. For this tutorial, we will use something similar to the following:

II. Structural Preparation

Preparation in Chimera

In this AMBER tutorial, we will use the same system with previous DOCK part. To begin with, we need three files under directory 001.CHIMERA.MOL.PREP.

Downloading the PDB file (1LOQ)

Since we need to edit the PDB before we use it in Chimera we should do this manually. Go to PDB homepage (http://www.rcsb.org/pdb/home/home.do ) enter the protein ID (1LOQ) in the search bar, click Download Files in the top-right of the webpage, then select PDB File (text). In the new window, save the file in Downloads.

Preparing the ligand and receptor in Chimera

In this section, we will create three new files and save them in the 001.CHIMERA.MOL.PREP/ folder:

1LOQ.dockprep.mol2 (complete system with hydrogens and charges)
1LOQ.receptor.noH.pdb (the receptor alone, without hydrogens)
1LOQ.ligand.mol2 (the ligand alone)

To prepare these files, first copy the original PDB file into the 001.CHIMERA.MOL.PREP/ folder and open it with VIM ($ vim 1LOQ.pdb). Because the residue name of the ligand (U) will give us some problems when assigning charges, change the residue name "U" to "LIG" starting at line 2082. Here is an example command that will change all instances of " U" into "LIG", while preserving the correct spacing:

:%s/U/LIG/gc

For this command, g is short for global and c is short for check with the user before making the change.

Next, open up the PDB file (1LOQ.pdb) in Chimera. To delete water molecules and other ligands, click Tools -> Structure Editing -> Dock Prep. Check all boxes and click Okay to the end. Alternatively, the waters can be deleted manually by choosing Select -> Residue -> HOH, then go to Actions -> Atoms/Bonds -> Delete. Hydrogen atoms can be added manually by choosing Tools -> Structure Editing -> Add H.

Next, to add charges to the ligand and receptor, go to Select -> Residue -> LIG, then go to Tools -> Structure Editing -> Add Charge. Choose AMBER ff99SB as the charge model, click Okay, and when prompted chose AM1-BCC charges for the ligand, and make sure the Net Charge is set to -1. (You must consider the chemistry of the ligand when assigning a charge state).

antechamber

The antechamber program itself is the main program of Antechamber package. In most of cases, one should use this program instead of a series of separated programs to do molecular format convertion, atom type assignment and charge generation etc. An antechamber input file requires all the atom names to be unique. So if we use 1LOQ.ligand.mol2 as the input file, it will cause errors. The program can only recognize atom names of 3 characters ( In this case, H5' and H5'' cannot be distinguished from each other. )

To begin with, go to 002.ANTE.TLEAP directory.
To make sure we have access to the three programs that we want to run (antechamber, parmchk and tleap) and we are using the correct version of amber, we can use the which command, type:

parmchk

Parmchk is another program in Antechamber. After running antechamber, we run parmchk to check the parameters. If there are missing parameters after antechamber is finished, the frcmod template generated by parmchk will help us in generating the needed parameters:

parmchk -i 1LOQ.lig.ante.prep -f prepi -o 1LOQ.lig.ante.frcmod

tleaP

Next, we need 3 new input files – “tleap.lig.in”, “tleap.rec.in” and “tleap.com.in”, for the ligand, the receptor, and the complex, respectively. These input files will be used by LEaP to create parameter/topology files and initial coordinate files.

Copy parameters of ions to your working directory from the following resource:

Visualization in VMD

Visualization is an important step in AMBER molecular dynamics simulation as it allows for the viewing of molecules and molecule movements within a specified field of view. Several files prepared in the previous two steps will be required for the visualization of the ligand and its movement in the protein binding pocket. The first step that must be completed is the copying of all necessary information from Seawulf to Herbie.

Then, when in Herbie, type the command vmd in any directory and the program VMD will open on the computer. In the "VMD Main" window, click File > New Molecule. Now in the new window titled "Molecule File Browser" a few files must be selected and loaded. To select and load a file follow these steps:

1. Click Browse... and select the file that you desire.
2. Click the down button in the "Determine file type:" field and select the proper file type.
3. Click Load and view the molecule/system in the original "VMD 1.8.5 OpenGL Display" window.

We can now visualize several files: the protein-ligand complex in the gas or water phase, and the ligand in the gas or water phase. Viewing the protein-ligand complex in the gas phase we select 1LOQ.com.gas.leap.prm7 and the file type AMBER7 Parm. Next you need to select and load the file 1LOQ.com.gas.leap.rst7 and the file type AMBER7 Restart. This can be done for the complex or ligand for either the gas or the water phase by selecting and loading the corresponding .parm7 files and .rst7 files.

Next, you can choose to edit the structure shown for better visualization. By clicking Graphics > Representations... a new window "Graphical Representations" will open, in which you can create new representations of the protein-ligand complex. It is possible to choose the entire complex or simply the protein or ligand, to color the structure in various ways, and to choose how it is best represented (e.g. lines, thicker bonds, full surface).

III. Simulation using sander

Minimization

Energy minimization is first performed on the stucture before the equilibration and production runs may be performed. Model building often creates unwanted structural artifacts that must be removed before a molecular dynamics simulation is performed.

To begin, create four files for minimization steps 1,3,4 and 5.

vim 01mi.in

and follow the naming according to minimization run number, i.e. 03mi.in

All "#mi.in" file content will be identical except the last parameter, the restraint weight (restraint_wt). This restraint will decrease with increasing minimization runs. Run number 1,3,4,and 5 has restraint weight 5,2,0.1,and 0.05 respectively.

ntmin:how many cycles will use a deepest decent method, the remaining cycles use an approximation of this called the conjugate gradient method.

ntx:only coordinates and not velocities are to be read from previous step

ntc:indicates level of constraint on bonds. if =1, SHAKE algorithm is off so no bonds are constrained. If =2, constrains any bonds with H atoms. If =3, constrains all bonds.

ntf:=1, all parts of the potential must be evaluated

ntb:periodic boundary to keep system at constant volume

ntp:=0, NO constant pressure applied

The frequencies at which the program records data are in controlled by the paramenters ntwx, ntwe, and ntpr.

ntwx:=1000, atom coordinates saved into .trj file every 1000 cycles

ntwe:=0, no .en file is generated

ntpr:=1000, energy readins are written as .out and .info files every 1000 steps

ntr:=1, positional restraint method applied

restraintmask= ':1-119 & !@H : position of atom within residues 1-119 that is not a H atom is being restrained

restraint_wt: restraint weight indicating how strong the restraint on the atoms is

Equilibration

To further prepare our complex for the molecular dynamic simulations, the subsequent step of energy minimization is equilibrate the system at some certain temperatures. We repeat the process of minimization and equilibration for twice in our case, of course with varied parameters and restraints put on our system.

Right after the first 1000 steps of minimization, we perform a 50000 steps(nstlim = 50000) X the step length of 1 fs(dt = 0.001) (that is 50 ps in total) equilibration at the temperature 298.15K(temp0 = 298.15, tempi = 298.15), contining putting on the weight (in kcal/mol) of 5.0 for the positional restraints on all non hydrogen atoms(restraintmask = ':1-210 & !@H=', restraint_wt = 5.0).

iwrap = 1 indicate that the coordinates written to the .rst7 and .trj files will be wrapped into a primary box. This reduces the amount of coordinate output, thus preventing overflowing and making visualization more convenient.

cut = 8.0 set the the non-bonded cutoff distance at 8.0 Angstroms.

Continuing 4 simulations are carried out after the fourth minimization, there are only little differents in these input files:

Note in the above script that for each run, the .rst7, .trj and .info files generated from the previous run provides the initial state to start from. The .prm7 file generated for the hydrated complex by TLEAP provides the force parameters.

Submit the file to the queue and monitor progress.

IV. Simulation Analysis

Ptraj

You should create another work directory for this step (004.PTRAJ, for example), if you don't have one.
There will be five runs of analysis, each of which will require different input files.

1. At first we want to concatenate the two 1ns trajectories together, stripping off the waters, and creating a .strip-file as an output. Below is the input file which will allow us to do so.

The two sets of numbers 1 1000 1 give the input information about which frames are used for the Ptraj. The first two numbers 1 and 1000 specify the starting and ending snapshots from the trajectory file. The ending number of the snapshot doesn't need to be accurate because if you actually don't have enough snapshots in your trajectory file, Ptraj will read up to the last one you have. The last number 1 specifies the frequency of the snapshot saved, in this case, we are saving every frame of the trajectory file. And the last line of the input file will take away all the water molecules.

As the input file is prepared we can launch the first analysis as follows:

Since we have just concatenated the two trajectories, we will have 2000 snapshots in 1LOQ.trj.strip. The third line in the input specifies the reference file, we have taken away all the water molecules during the first step, hence we are working here with the gas phase complex. The last line says we are calculating the rmsd for alpha carbon number 1 to 209.

By doing this we will compare the trajectory file to 1LOQ.com.gas.leap.rst7 as well, but working with the ligand instead of the receptor (we specified that by pointing that we want to calculate the rmsd for carbon, nitrogen, oxygen and sulfur in residue 210.

The last two steps are to obtain energetic information about the system. To do this we take a trajectory file of the gas phase complex 1LOQ.com.trj.stripfit, and want to create two more trajectory files containing the information on only receptor and only ligand correspondingly.

4. At this step we consider receptor only. The input file is provided below:

As we've gone through all these steps, the analysis is done. If you want to visualize the trajectories, you first need to copy the trajectory files to Herbie like this, for example (being a level above 004.PTRAJ directory):

Now, launch VMD, then open one of the .prm7 files in 002.ANTE.TLEAP. If you want to visualize the whole complex in gas state, you can open 1LOQ.com.gas.leap.prm7 with AMBER7 Parm from 002.ANTE.TLEAP and then 1LOQ.com.trj.stripfit with AMBER coordinates from 004.PTRAJ. With these files, you can look at the real-time movement of the complex in the gas state. You can repeat this procedure to observe the real-time movement of the complex in the water state. Just open 1LOQ.com.wat.leap.prm7 instead of 1LOQ.com.gas.leap.prm7.

RMSD Plots

Once having finished running Ptraj, you should find two RMSD files in your directory: 1LOQ.lig.rmsd.txt and 1LOQ.rmsd.CA.txt
The data in these files can be plotted to visualize the structural drift (ie change in RMSD) over the course of your simulation. Large fluctuations in RMSD may be an indication that the simulation was unstable.

Here is a plot for ligand RMSD:

Measuring h-bond distances

MM-GBSA Energy Calculation

MM/GBSA is the acronym for Molecular Mechanics/Generalized Born Surface Area. This part of AMBER combines molecular mechanics (MM) with both the electrostatic (PB) and nonpolar (SA) contribution to solvation . Topology files are needed for the receptor, ligand, and receptor-ligand complex. The trajectory files generate the coordinates. Therefore, molecular dynamics is used to generate a set of snapshots taken at fixed intervals from the trajectories. These snapshots are processed to remove solvent and generate the free energy of binding.

Then this script should be sent to the queue, i.e., qsub the script using the commands:

qsub run.sander.rescore.csh

You can monitor your progress by typing

qstat -u username

When the job is complete, you will obtain the following output files: gb.rescore.out.com, gb.rescore.out.lig, and gb.rescore.out.rec
In these files, the single point energy calculation results will be written for each individual frame. It will be found in the results section and the output file will have an infrastrucutre that is similar to the following:

You can take these text files, import them into Excel, and do the rest of your modifications there.

Equations for analysis

Remember that to obtain the Gvdw term, you need to take the SASA (which is ESURF) and input into equation 1:

ΔGnonpolar = SASA*0.00542 + 0.92

Also, the mmgbsa of a given system can be determined by equation 2:

ΔGmmgbsa = ΔGvdw + ΔGcoul + ΔGpolar + ΔGnonpolar

From the output file:

VDWAALS = ΔGvdw

EELS = ΔGcoul

EGB = ΔGpolar

You can then easily calculate the ΔΔGbind by using equation 3:

ΔΔGbind = ΔGmmgbsa,complex – (ΔGmmgbsa,lig + ΔGmmgbsa,rec)
You will want to careful when doing your analysis that the results from frame 1 for the receptor and ligand are subtracted from the results from frame 1 for your complex. By doing this in excel, you should have 2000 frames for each, and the values should cleanly line up. Finally, you will want to plot your ΔΔGbind and examine if you see corresponding changes in the ligand position and the ΔΔGbind. Also, you should determine the mean and standard deviation for your ΔΔGbind.

Plotting Energy

When your rescoring calculation finishes, you have the following three output files: "gb.rescore.out.com", "gb.rescore.out.lig", and "gb.rescore.out.rec".

Use the following script, entitled get.mmgbsa.bash, to extract data and calculate MMGBSA energy for each snap shot.